MiRNA-mRNA Association Analysis Of DHAV-1 Infected DEF And Apl-miR-425-3p Targeting SEC22B To Promote Viral Replication

Duck hepatitis A type 1(DHAV-1)infection can cause viral hepatitis in ducklings,which is characterized by rapid onset,short duration,rapid transmission and high mortality,and has seriously hampered the development of the duck industry in China.In order to better elucidate the mechanism of DHAV-1-host interaction,this paper used high-throughput sequencing technology to obtain micro RNA(miRNA)and mRNA expression profiles after DHAV-1infection in duck embryo fibroblast(DEF)cells,and performed association analysis of the differential expressed miRNA,potential DHAV-1 viral small RNA(vs RNA)and differentially expressed mRNA,the DHAV-1 genome based on the targeting regulatory relationship between miRNA and mRNA.Finally,a preliminary investigation of miRNA-apl-miR-425-3p and its mechanism affecting viral life activity was conducted,and the results are as follows:1.miRNA-mRNA association analysis of DHAV-1-infected DEFDEF was infected with DHAV-1 at MOI=1 splice dose,the cytopathic lesion of each group were observed at different time points and the viral load was detected.The group that have reached peak viral load(48 h post-infection)were selected for high-throughput sequencing according to the results above.The expression of 65 miRNA and 2297 mRNA were significantly changed in the virus-infected group,of which 35 miRNA and 1356 mRNA were significantly up-regulated and 30 miRNA and 941 mRNA were significantly down-regulated;31 potential DHAV-1 vs RNA were obtained by comparing the high-throughput sequencing data with the DHAV-1 genomic sequence.Seven differentially expressed miRNA,four potential DHAV-1 vs RNA and nine differentially expressed mRNA were selected for validation,and the results were generally consistent with the high-throughput sequencing results.Four targeted relationship regulatory networks were further constructed using differentially expressed miRNA,differentially expressed mRNA,potential DHAV-1 vs RNA and DHAV-1 genomic.Further analysis revealed that the up-regulated miRNA in differentially expressed miRNAdifferentially expressed mRNA regulatory network were mainly involved in regulating environmental information processing-related pathways such as MAPK and neural ligandreceptor interactions,and the down-regulated miRNA were mainly involved in regulating herpes simplex virus infection pathway and some metabolism-related pathways such as glycosphingolipid biosynthesis;the miRNA in DHAV-1 vs RNA-differentially expressed mRNA regulatory network are mainly involved in regulating environmental information processing-related pathways such as neuroactive ligand-receptor interactions,calcium ion signaling and focal adhesion;DHAV-1 structural proteins VP0 and VP1 in differentially expressed miRNA-DHAV-1 genomic regulatory network had the highest number of differentially expressed miRNA targeting relationships;VP0 in DHAV-1 vs RNA-DHAV-1genomic regulatory network had the highest number of DHAV-1 vs RNA targeting relationships.These suggested that environmental information processing-related pathways and VP0 are important pathways for DHAV-1-host interactions.2.apl-miR-425-3p affects DHAV-1 replication by targeting and regulating SEC22 B expressionapl-miR-425-3p,which targets molecules in the MAPK pathway,apl-miR-130b-5p,which targets VP0,and two differentially expressed miRNA(apl-miR-1454,apl-miR-1434)were selected to initially investigate their effects on DHAV-1 replication and the mechanisms involved it.The mimics of the four miRNA were transfected into DEF cells followed by infection with DHAV-1.The results showed that overexpression apl-miR-130b-5p and apl-miR-425-3p significantly increased the viral load in cells.Subsequently,the target gene binding sites of apl-miR-425-3p and apl-miR-130b-5p in the host genome and viral genome were predicted using miRanda and RNAhybird software and the targeting relationships were verified by dual luciferase reporter system.It was found that apl-miR-425-3p targets MRAS and SEC22 B,and apl-miR-130b-5p did not target VP0 and SEC23 B.Furthermore,the effect of overexpression of apl-miR-425-3p on the transcription and expression of target gene were further detected by RTq PCR and WB.The results suggested that overexpression of apl-miR-425-3p significantly reduced the transcription and protein expression of SEC22 B but had no effect on the transcript level of MRAS,which indicated that apl-miR-425-3p could target and regulate the expression of SEC22 B.Finally,eukaryotic expression plasmids of SEC22 B were constructed and transfected into DEF cells followed by infection with DHAV-1,the viral load and the expression of viral protein VP3 in each group were detected by RT-q PCR and WB.The results showed that overexpression of SEC22 B significantly reduced the viral load and the expression of VP3.In summary,these results indicated that apl-miR-425-3p could promote DHAV-1 replication by targeting and regulating SEC22B expression.